CN114891809B - 谷胱甘肽s转移酶基因在提高芒果维生素c含量中的应用 - Google Patents
谷胱甘肽s转移酶基因在提高芒果维生素c含量中的应用 Download PDFInfo
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Abstract
本发明属于分子育种技术领域，公开了一种谷胱甘肽S转移酶基因MiGST1在提高芒果维生素C含量中的应用。所述基因MiGST1的核苷酸序列如SEQIDNO：1所示。本发明通过QTL定位、基因注释和代谢合成途径分析表明，编码谷胱甘肽S转移酶(GST)的MiGST1基因为抗坏血酸生物合成相关基因，MiGST1可以显著提高芒果果实抗坏血酸含量。GST酶活性检测表明，抗坏血酸含量随着谷胱甘肽S转移酶活性的增强而增加。通过分析谷胱甘肽S转移酶活性、基因表达和基因结构进一步验证了结果。这些发现阐明芒果中抗坏血酸的复杂调控机制。
Description
技术领域
本发明属于分子育种技术领域，具体涉及一种谷胱甘肽S转移酶基因在提高芒果维生素C含量中的应用。
背景技术
芒果是多年生木本植物，幼年期长，基因组复杂且杂合度高，难以构建高世代自交系或回交种群。Hemmat等人(1994)提出了“双测试杂交”理论来解决这个问题。Kashkush等人(2001)利用AFLP构建了芒果的第一个初步分子遗传连锁图谱，由13个连锁群组成，总遗传距离为161.5cM，由13个连锁群组成。Khalil等人(2001)重新分析了作图群体，利用15个AFLP引物扩增了该种群的60株F1植株，获得了191个多态位点，构建了15个连锁群，总遗传距离为354.1cM。Chunwongse等人(Molecular mapping of mango cultivars‘Alphonso’and‘Palmer’.ISHS Acta Horticulturae 509:VI International Symposium onMango.Doi:10.17660/ActaHortic.2000.509.19)使用来自‘Alphonso’和‘Palmer’杂交的31个F1后代构建了一个带有RFLP标记的遗传连锁图谱，总遗传距离为529.9cM。
随着测序技术的发展，基于高通量测序技术的SNP标记被广泛应用于许多果树遗传连锁图谱的构建中。基于高通量测序，Luo等人(Construction of a high densitygenetic map based on large scale marker development in mango using specificlocus amplified fragment sequencing(SLAF-seq).Frontiers in Plant Science 7:1310)基于SLAF-seq构建了芒果高密度整合遗传图谱，包括20个连锁群(LGs)，图谱总长度为3,148.28cM，标记间平均距离为0.48cM。Kuhn等人(Genetic Map of Mango:A Tool forMango Breeding.Frontiers in Plant Science 8:577)使用芒果中的729个SNP标记开发了一个共有的遗传连锁图谱。
果实品质包括物理和化学性质，物理性质包括果实的形状、大小、皮肤的光滑度和颜色等；化学性质涵盖可食性、糖、酸、维生素、氨基酸、蛋白质、固体、类胡萝卜素等。抗坏血酸(AsA)是水果最重要的质量参数之一，尽管已经对维生素的含量进行了许多研究，但芒果果肉中的维生素C的分子调控机制及标记辅助育种(或分子育种)尚未见报道。
随着人们生活水平的提高，芒果果实品质的提高变得越来越重要。育种者早就认识到作物品质与产量显着负相关。虽然传统育种在芒果新品种的开发中发挥了重要作用，但芒果树童期长，这些目标性状受多个位点控制，严重阻碍了芒果果实品质育种的进程，取得的成果和进展相当缓慢。分子标记的发展使分子标记辅助选择在作物育种中成为可能，大大缩短了育种时间，提高了育种的精度。
提高果实品质是芒果育种计划的重要目标。实践证明，培育和推广品质优良的芒果新品种，是提升全球芒果产业竞争力最经济、最有效的途径。因此，迫切需要加强芒果果实品质优良基因的发现和研究，尤其是果实性状的糖质和有机酸品质。果实品质性状的分子标记辅助育种在许多果树上已经取得很大进展，举一些例子，张等人(Identification,characterization,and utilization of genome-wide simple sequence repeats toidentify a QTL for acidity in apple.BMC Genomics 13:537)利用310个SSR标记构建了苹果遗传连锁图谱，在连锁群8上定位了一个控制苹果酸性性状的显性QTL，可以解释13.8％的表型变异，可用于分子标记辅助选择。Yamamoto等(Identification of QTLscontrolling harvest time and fruit skin color in Japanese pear(Pyruspyrifolia Nakai).Breeding Science 64:351-361)构建了包含278个位点、长度为1039cM的遗传连锁图谱，并在LG8连锁群上绘制了一个控制果皮颜色的显性QTL，可连续两年在多个位点检测到，可用于分子生物学研究。吴等人(High-density genetic linkage mapconstruction and identification of fruit-related QTLs in pear using SNP andSSR markers.Journal of Experimental Botany 65(20):5771-5781)利用构建的梨遗传连锁图谱定位可溶性固形物含量、果皮颜色和果皮光滑度的QTL，这些QTL首次被检测到，对分析梨果实品质性状的分子遗传机制具有重要意义；Verdu等人(QTL Analysis andCandidate Gene Mapping for the Polyphenol Content in Cider Apple.PLoS ONE 9(10):e107103)利用构建的苹果遗传连锁图谱检测多酚的主要QTL，可用于分子标记辅助育种以改良苹果品种。Alpert和Tanksley(fw 2.2:a major QTL controlling fruit weightis common to both red-and green-fruited tomato species.Theor Appl Genet 91:994-1000)检测到番茄果实重量的主效QTL，fw2.2，它位于2号染色体上，解释了F2近同基因系(NIL)种群中总表型变异的30％-47％。幸运的是，Liu等人(Generation and Analysisof an Artificial Gene Dosage Series in Tomato to Study the Mechanisms byWhich the Cloned Quantitative Trait Locus fw2.2 Controls Fruit Size.PlantPhysiology 132(1):292-299)克隆了QTL fw2.2并对其进行了功能鉴定，fw2.2的克隆使人们对果实发育的分子机制和果实大小特征的基因工程有了更好的了解。Kuhn等人(GeneticMap of Mango:A Tool for Mango Breeding.Frontiers in Plant Science 8:577)使用7个F1杂种群体开发了一个共有的遗传连锁图谱，鉴定了14个表型性状，其中一个非常重要的性状多胚性被定位到芒果的连锁群8上。迄今为止，在芒果中构建了四个遗传连锁图谱。果实品质性状的分子标记辅助育种已在苹果、梨等多种果树上成功应用。抗坏血酸是决定芒果果实或其它作物营养品质的主要成分。专利CN112080509A公开了一种草莓维生素C的合成相关基因FaGalLDH，将该基因在拟南芥中超量表达后，得到维生素C(AsA)含量显著提高的转基因植株，且提高了转基因植株的耐盐性。实验证明，FaGalLDH基因可以在体外合成维生素C，并且优化发现了最佳诱导条件，超量表达可显著提高转基因拟南芥叶片的维生素C含量，且对植物的正常生长没有明显的影响。专利CN 104372015 B公开了一种花生维生素C合成相关基因AhPMM，将该基因在花生中超量表达后，得到总维生素C和还原态维生素C(AsA)含量显著提高的转基因植株。实验证明，AhPMM基因超量表达可显著提高花生叶片的维生素C含量，且对花生的正常生长没有明显的影响。然而，芒果中维生素C的QTL定位和维生素C合成的分子机制尚未见报道。
发明内容
针对以上现有技术存在的缺点和不足之处，本发明的首要目的在于提供一种谷胱甘肽S转移酶基因MiGST1(gene ID:mango032916)。
本发明的另一目的在于提供上述谷胱甘肽S转移酶基因MiGST1在提高芒果维生素C含量中的应用。
本发明的再一目的在于提供谷胱甘肽S转移酶在提高芒果维生素C含量中的应用。
本发明目的通过以下技术方案实现：
一种谷胱甘肽S转移酶基因MiGST1，所述基因MiGST1的核苷酸序列如SEQ ID NO：1所示。
上述谷胱甘肽S转移酶基因MiGST1在提高芒果维生素C含量中的应用。
谷胱甘肽S转移酶在提高芒果维生素C含量中的应用。
与现有技术相比，本发明的有益效果是：
(1)本发明通过基因注释和代谢合成途径分析表明，编码谷胱甘肽S转移酶(GST)的MiGST1基因，为抗坏血酸生物合成相关基因，位于qAA1的定位区间。GST酶活性检测表明，抗坏血酸含量随着谷胱甘肽S转移酶活性的增强而增加。
(2)本发明通过qRT-PCR结果证明基因MiGST1在“Irwin”中表达，但在“Jin-Hwang”中不表达。为了解析“Jin-Hwang”和“Irwin”之间的MiGST1在两种材料中表达不同的分子机制，在“Jin-Hwang”和“Irwin”之间进行了全基因组重测序。结果表明，“Jin-Hwang”中存在终止密码子突变，导致终止密码子丢失，从而导致“Jin-Hwang”中该基因失活。因此，我们最终确定了MiGST1作为候选基因，编码谷胱甘肽S转移酶调节芒果抗坏血酸(维生素C)含量。
附图说明
图1～3分别为2019～2021年“Jin-Hwang”דIrwin”作图群体果实中抗坏血酸含量的分布图。
图4为在高密度整合遗传连锁图谱上鉴定抗坏血酸含量的QTL结果图。图中A为沿20条染色体的抗坏血酸性状变异的LOD值；B为1号染色体上抗坏血酸含量变化的LOD值。
图5为基于AsA和GSH的抗氧化系统的假定代谢相互作用图。
图6为在亲本(“Jin-Hwang”和“Irwin”)和F1代中检测到的GST酶活性变化规律图。
图7为qRT-PCR分析确定不同抗坏血酸含量的“Jin-hwang”和“Irwin”果肉中MiGST1基因的表达结果图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例
1材料和方法
1.1群体作图
在本研究中使用了在“Jin-Hwang”(母本)和“Irwin”(父本)之间杂交的173个分离的F1种群进行基因分型。这些植株生长在中国热带农业科学院南亚热带作物研究所。对于亲本，“Jin-Hwang”和“Irwin”的抗坏血酸含量差异显着，“Irwin”的抗坏血酸含量高于“Jin-Hwang”(见表1)。
1.2大田种植与性状检测
2019年、2020年和2021年，在广东省湛江市(21°27'N，110°32'E)的热带水果国家田间基因库中种植了由173个杂交后代组成的F1作图群体的田间试验。两个亲本“Jin-Hwang”和“Irwin”也被包括在内作为该领域的对照。
1.3果肉样品和抗坏血酸含量的测量
成熟时收集了173个杂交后代和两个亲本的果实，通过检查果皮颜色并确认果肉颜色变为淡黄色来评估成熟度。将这些水果置于25℃、81％-95％RH下，直至它们自然成熟。173个杂种后代和两个亲本各制备10个果实；沿果腹缝合线的上、中、下部分收集果肉，切成小块。将纸浆样品(三个重复)用液氮快速冷冻并储存在-80℃冰箱中直至使用。通过高效液相色谱法(HPLC)测量抗坏血酸含量。
1.4遗传图谱构建和QTL分析
我们实验室使用6,594个SNP标记构建了高密度遗传图谱，R/qtl软件中的区间作图(IM)模型用于QTL定位。通过1000个排列估计LOD阈值以确定显著QTL。
1.5谷胱甘肽S转移酶(GST)活性的测量
谷胱甘肽S转移酶(GST)试剂盒购自苏州格雷斯生物科技有限公司(中国苏州)，根据制造商的使用说明测量GST活性。
1.6候选基因的表达分析
使用PrimeScriptTMRT试剂盒将芒果果实的三个生物复制品的总RNA转化为cDNA。采用Primer 5.0软件设计qRT-PCR引物序列。芒果肌动蛋白被用作参考基因。qRT-PCR使用
480 Real-Time PCR System(Roche，Germany)和SYBR Green II PCR MasterMix(Takara，Japan)，根据制造商的使用说明进行。
1.7候选基因的全基因组重测序分析
“Jin-Hwang”和“Irwin”的全基因组重测序和数据分析由中国南京的集思慧远生物技术有限公司完成。
1.8数据分析
使用IBM SPSS Statistics 26对果实品质性状数据进行统计分析。
2结果
2.1 F1群体和两个亲本抗坏血酸含量的评价
F1群体和两个亲本抗坏血酸含量的实测数据如表1所示。可见“Jin-Hwang”和“Irwin”抗坏血酸含量差异显着，“Irwin”抗坏血酸含量高于“Jin-Hwang”。F1群体中抗坏血酸含量的值分布在两个父母之间。偏度和峰度值的结果表明，抗坏血酸含量符合正态分布，并在F1群体中表达了两个方向的连续分离(如图1～3所示)。
表1 F1和两个亲本抗坏血酸含量统计分析(mg/100g FW)
2.2抗坏血酸含量的遗传连锁图构建和QTL定位
利用6594个SNP标记构建了高密度的综合遗传连锁图谱，由20个连锁群组成，总长度为3148.28cm，标记之间的平均距离为0.48cm。
由于抗坏血酸含量在F1代是连续变化的，符合正态分布，因此采用R/qtl软件中的IM模型进行QTL作图，并采用scanone函数进行计算。对抗坏血酸含量进行了QTL定位，在1号染色体上鉴定出一个主效QTL qAA1(LOD>6.0)(如表2和图4所示)，它解释了24.68％-26.00％的表型变异，位于‘Tommy-Atkins’参考基因组1号染色体上，介于Marker67211和Marker26426之间，跨越约0.28cM的遗传距离。
表2“Jin-Hwang”דIrwin”作图群体中抗坏血酸含量的QTL鉴定
2.3位点分析和候选基因预测
通过对“Tommy-Atkins”参考基因组(https://www.ncbi.nlm.nih.gov/assembly/GCA_016746415.1)的比对揭示了qAA19中的八个预测蛋白质编码基因间隔(长度约为0.28Mb，带有10个SLAF标记)。在Swiss-Prot和BLASTX分析之后，所有这些基因都被鉴定和注释(如表3所示)。
表3 1号染色体基因定位区域内候选基因的预测
为了更有效地筛选候选基因，有必要缩小抗坏血酸的候选基因范围，根据芒果参考基因组的基因注释信息，鉴定出8个候选基因。这八个基因的表达水平通过两个亲本之间的qRT-PCR分析进一步验证。除了候选基因mango032916之外，其余候选基因在两个父母之间的qRT-PCR结果相似，该候选基因因为位于第1号染色体命名为MiGST1，其核苷酸序列如SEQ ID NO：1所示。基于AsA和GSH的抗氧化系统的假定代谢相互作用图如图5所示。根据表3和图5中基因注释和代谢合成途径分析表明，编码谷胱甘肽S转移酶(GST)的MiGST1，使其成为抗坏血酸生物合成相关基因。
2.4候选基因的进一步验证
为初步验证谷胱甘肽S转移酶与抗坏血酸的关系，采用谷胱甘肽S转移酶试剂盒检测两个亲本(“Jin-Hwang”和“Irwin”)和杂种F1群体中谷胱甘肽S转移酶的酶活性。结果如图6所示，结果表明，两个父母之间的谷胱甘肽S转移酶活性存在显着差异。“Irwin”的谷胱甘肽S转移酶活性显着高于“Jin-Hwang”，“Jin-Hwang”和“Irwin”的杂种F1群体杂交也显示出相同的趋势。抗坏血酸含量随着谷胱甘肽S转移酶活性的增强而增加，这些结果表明抗坏血酸含量与谷胱甘肽S转移酶活性显着相关。
为了进一步验证与抗坏血酸相关的候选基因MiGST1，对两个亲本进行了qRT-PCR分析；芒果肌动蛋白被用作参考基因。qRT-PCR的引物由Primer Premier 5.0设计，正向引物为ATTGCTGCCTTCACATCC，反向引物为TTGCTGCTTCCTTCACTTC。有趣的是，qRT-PCR结果证明基因MiGST1在“Irwin”中表达，但在“Jin-Hwang”中不表达，在F1杂交后代中，基因MiGST1的表达量随着抗坏血酸含量的增大而增强(如图7所示)。
为了解决“Jin-Hwang”(金煌)和“Irwin”(爱文)之间的MiGST1在两种材料中表达不同的分子机制，基于“Jin-Hwang”和“Irwin”之间的重测序进行基因差异序列分析。结果表明，在“Jin-Hwang”和“Irwin”之间，该基因MiGST1有114个SNP或InDel突变，其中113个突变为同义突变，没有改变基因的功能。特别值得注意的是，“Jin-Hwang”中终止密码子的突变导致终止密码子丢失，从而导致“Jin-Hwang”中该基因失活(如表4所示)。这一结果也从碱基序列的角度验证了qRT-PCR在“Jin-Hwang”中没有表达的原因。
表4全基因组重测序基因MiGST1在“金煌”和“爱文”之间的变异分析
在这项研究中，MiGST1作为谷胱甘肽S-转移酶的候选基因已被证明与抗坏血酸含量有关。因此，该基因或由该基因表达的谷胱甘肽S-转移酶的应用可以促进芒果果实中抗坏血酸含量提高的分子育种。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其它的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
序列表
<110> 中国热带农业科学院南亚热带作物研究所
<120> 谷胱甘肽S转移酶基因在提高芒果维生素C含量中的应用
<130> 2022-4-25
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Claims (2)
1.一种谷胱甘肽S转移酶基因MiGST1在提高芒果维生素C含量中的应用，其特征在于，所述基因MiGST1的核苷酸序列如SEQ ID NO：1所示。
2.一种由基因MiGST1编码的谷胱甘肽S转移酶在提高芒果维生素C含量中的应用，其特征在于，所述基因MiGST1的核苷酸序列如SEQ ID NO：1所示。
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